Anti mouse antibody

Cells at a concentration of 1 × 10⁵ cells/mL were plated into a 6-well dish. After 3 days of incubation, cells in each well were lysed with lysis buffer at 4 °C for 30 min. The lysates were loaded and analyzed using SDS-PAGE following standard protocols. Primary antibodies used were anti-REG4 antibody (1:500, Abcam, Cambridge, UK) and anti-Actin antibody (1:1000, Proteintech, Wuhan, China). Secondary antibodies used were anti-mouse antibodies (1:1000, Proteintech, Wuhan, China) and anti-rabbit antibodies (1:1000, Proteintech, Wuhan, China).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), TRIzol and Lipofectamine™ 2000 reagents were purchased from Invitrogen (Camarillo, CA, USA). Rg3 was provided by Dalian Fusheng Pharmaceutical Co., Ltd. (Dalian, China). It was diluted with cell culture media to final concentration. Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Annexin V/FITC kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, Jiangsu, China). Antibodies specific for PARP, caspase-3, -8, -9, Bcl-2, survivin, Bax, FUT4, β-actin, HRP-conjugated anti-rabbit and anti-mouse antibodies were purchased from Proteintech Group (Wuhan, China). Antibodies specific for p-p65, p65, pIκBα, IκBα, pIKKα/β and IKKα/β were purchased from Cell Signaling Technology (Boston, MA, USA). p65 siRNA was purchased from the Shanghai GenePharma Co. (Shanghai, China). NF-κB inhibitor (Bay 11-7082) was purchased from Selleck Chemicals (Houston, TX, USA). Nuclear extract kit and EMSA kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ChIP kit was purchased from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) assay kit was purchased from Amersham Biosciences (Pittsburgh, PA, USA).

Cells were washed with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl, 1% NP-40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EDTA) containing 1 mM PMSF and 1x protease inhibitor (539134, Calbiochem). Total cell lysates were then collected by sonication of the cells followed by centrifugation at 16,000g for 5 min at 4 °C. Total cell lysates (30–50 μg) were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were hybridized with anti-p53 (Proteintech Group), anti-p53-S15 (Cell Signaling Technology), p53-T18 (GeneTex, Inc), p53-T55 (Abcam), p53-T81 (Cell Signaling Technology), p53-T155 (Santa Cruz Biotechnology, Inc), anti-Cdc25B (Cell Signaling Technology), anti-Cdk1 (Santa Cruz Biotechnology, Inc), anti-pCdk1-Y15 (Abcam), anti-p27 (GeneTex, Inc), anti-p21 (Cell Signaling Technology), anti-p16 (Proteintech Group), anti-pRB (S780, Cell Signaling Technology), or anti-RB (Proteintech Group). Horseradish-peroxidase-conjugated donkey anti-rabbit (GE Healthcare Life Sciences) or anti-mouse antibodies (Proteintech Group) were used as the secondary antibodies. The T-Pro LumiLong Plus Chemiluminescent Substrate Kit (T-Pro Biotechnology) was used to visualize the antibody-bound proteins.